We report the challenges and resolutions facing the use of Chlorella protothecoides for metal bioavailability and toxicity research under acidic conditions. In our study, we found that a simplified growth medium recommended for metal bioavailability research did not support the optimum growth of C. protothecoides at pH 5.5, possibly due to nutrient deficiencies, rendering it unsuitable for bioavailability studies under acidic conditions. On the other hand, a modified Woods Hole MBL growth medium supplying all macro and micro nutrients, with chelators Tris and EDTA removed, and (NH4)2Fe(SO4)2 supplemented as the source of iron, produced excellent growth of the algae under identical conditions. This medium is therefore recommended as the alternative growth medium. While a pH increase of up to 2 units a day was observed in unbuffered media during algal growth, incorporation of CO2 into the aeration stream at a fixed partial pressure stabilized the medium pH. By initiating the metal (e.g. Cu) treatment at a culture optical density (750 nm) of 0.04, where the algal population was just entering the rapid growth phase, the Chlorella assay was completed within 48 h. This minimized metal speciation changes and nutrient limitation that may occur over longer test durations. As demonstrated with the case of Cu, a cell density of 7.5 x 105 cells mL–1 at the time of metal addition, caused only 5% initial loss of solution Cu, addressing the concern that such an initial cell density might deplete the metal from solution as a result of metal binding to algal cells. Overall, this study provides a valid means of employing Chlorella for metal bioavailability and toxicity research under acidic conditions, which ensures that algal growth is not impeded by nutrient deficiency, experimental duration is short, and more importantly, solution pH and metal speciation do not change significantly over the experimental period.